The invention relates to the modulation of human mast cell activation by compounds which modulate adenosine 5xe2x80x2-triphosphate (ATP) binding to ATP receptors (P2-purinoceptors) on the cells. The invention further relates to the treatment of disorders characterized by undesirable mediator release from stimulated mast cells, particularly immunologically stimulated lung mast cells. The invention also relates to methods for in vitro screening of candidate therapeutic agents for treating such disorders.
Mast Cells
Mast cells comprise a normal component of the connective tissue that plays an important role in immediate (type I) hypersensitivity and inflammatory reactions by secreting a large variety of chemical mediators from storage sites in their granules upon stimulation. Mast cells, and their circulating counterparts the basophils, possess surface receptors known as FCxcex5RI. The receptors are specific for antibody xcex5 heavy chains.
The event that initiates immediate hypersensitivity is the binding of antigen to IgE on the mast cell or basophil surface. Both cell types are activated by cross-linking of Fcxcex5RI molecules, which is thought to occur by binding multivalent antigens to the attached IgE molecules.
Mast cells may also be activated by mechanisms other than cross-linking Fcxcex5RI, such as in response to mononuclear phagocyte-derived chemocytokines, to T cell-derived cytokines and to complement-derived anaphylatoxins. Mast cells may also be recruited and activated by other inflammatory cells or by neurotransmitters which serves as links to the nervous system.
When antigen binds to IgE molecules attached to the surface of mast cells, a variety of mediators are released which give rise to increased vascular permeation, vasodilation, bronchial and visceral smooth muscle contraction, and local inflammation. In the most extreme form of immediate hypersensitivity reaction known as anaphylaxis, mediators released from mast cells can restrict airways to the point of asphyxiation. So-called atopic individuals, who are prone to develop strong immediate hypersensitivity responses, may suffer from asthma, hay fever or chronic eczema. These individuals possess higher than average plasma IgE levels.
Antigens that elicit strong immediate hypersensitivity reactions are known as allergens. Allergy afflicts twenty percent of the United States population.
Immediate hypersensitivity results from the following sequence of events: production of IgE by B cells in response to antigen, binding of the IgE to Fcxcex5RI on the surface of mast cells, interaction of re-introduced antigen with the bound IgE and activation of the mast cells and release of mediators. Antigen binding can be simulated by polyvalent anti-IgE or by anti-Fcxcex5RI antibodies. Such antibodies can activate mast cells from atopic as well as non-atopic individuals, whereas allergens activate mast cells only in atopic persons.
Mediators released from mast cells may be divided into two broad classes, pre-formed or secretory granule associated mediators and nonpreformed or newly synthesized mediators. The pre-formed mediators include biogenic amines, most notably histamine. The pre-formed mediators also comprise granule macromolecules such as proteoglycans, most notably heparin and chondroitin sulfate E; chemotactic factors such as eosinophil and neutrophil chemotactic factors of anaphylaxis; and enzymes such as proteases, tryptase, chymase, cathepsin G-like enzyme, elastase, carboxypeptidase A and acid hydrolases. The nonpreformed mediators include products of arachidonic acid, prostaglandin D2, leukotrienes C4 and B4 and platelet activating factor. Another class of mediators, the cytokines, are produced by mast cells upon IgE-mediated activation, or by other cells, including recruited TH2 lymphocytes. The cytokines are predominantly responsible for the late phase reaction which begins two to four hours after elicitation of many immediate hypersensitivity reactions. One cytokine, tumor necrosis factor alpha, may exist in the mast cells as preformed stores, or may represent a newly synthesized product released over a period of hours.
Mediators released from human mast cells are central to the pathophysiology of allergy, asthma and anaphylaxis. In particular, mast cells and their release of histamine and other mediators play an important role in the symptomatology of asthma and other human diseases. During the early phase of human lung hypersensitivity reactions upon exposure to antigen (i.e., pollens, cats, etc.), mast cells release and are the major source of histamine, and newly synthesized lipid products of arachidonic acid metabolism: prostaglandin D2 and leukotriene C4. These mediators produce immediate breathlessness, which subsides in one hour but returns within 2-4 hours (the xe2x80x9clate phasexe2x80x9d response). Attesting to their primal role in hypersensitivity responses, human lung mast cells (HLMC) are characterized by mRNA generation, protein synthesis and release of so-called TH2 cytokines within these first few hours of activation. These cytokines including IL-5, and IL-13 are believed to be central to the evolution of chronic allergic/asthmatic states. In the lung, only mast cells are a source of histamine. Thus, histamine release is a distinct marker of mast cell activation and behavior. For a review of the role of mast cells in inflammatory responses in the lung, see Schulman, Critical Reviews in Immunology, 13(1):35-70 (1993), the entire disclosure of which is incorporated herein by reference.
Clinically, asthma is recognized by airway hyperactivity and reversible airways obstruction. Pathological derangements at the tissue level include constriction of airway smooth muscle, increased vascular permeability resulting in edema of airways, outpouring of mucus from goblet cells and mucus glands, parasympathetic nervous system activation, denudation of airway epithelial lining cells, and influx of inflammatory cells. Underlying these tissue effects are direct effects of potent mediators secreted following physical, inflammatory, or immunological activation and degranulation. The early phase of the asthmatic reaction is mediated by histamine and other mast cell mediators that induce rapid effects on target organs, particularly smooth muscle. The pathophysiologic sequence of asthma may be initiated by mast cell activation in response to allergen binding to IgE. Evidence exists to link exercise-induced asthma and so-called xe2x80x9caspirin-sensitivexe2x80x9d asthma to HLMC degranulation. ps Pharmacologic Modulation of Mast Cell Function
A limited number of pharmacologic agents have been tested for effect on HLMC activation-secretion. The beta-adrenergic agonist pharmacologic agents, as typified by fenoterol, are the most potent global inhibitors of HLMC. Though widely touted as xe2x80x9cmast cell stabilizers,xe2x80x9d disodium cromoglycate and nedocromil sodium poorly inhibit purified HLMC histamine release. While certain corticosteroids have been found to suppress IgE-mediated generation of late-phase cytokine mRNA and protein (e.g., IL-5), release of early phase mediators (e.g., histamine and LTC4) are unaffected by corticosteroids. HLMC release has been shown to be inhibited by the immunosuppressant agents FK-506, cyclosporin A and auranofin. Arachidonate pathway inhibitors are of considerable importance, they may leave the release of other allergic mediators (e. g., histamine, proteases) unaffected. Such arachidonate pathway inhibitors include inhibitors of 5-lipoxygenase and inhibitors of cyclooxygenase.
Adenosine and Adenosine Triphosphate
ATP is found in every cell of the human body; it plays a major role in cellular metabolism and energetics. ATP is released into the extracellular fluid under physiologic and pathophysiologic conditions. For example, ATP is released from ischemic cells, activated platelets, apoptotic and necrotic cells, nerve terminals as a co-transmitter, and muscle fibers during exercise. Inhalation of aerosolized ATP has been shown to trigger bronchoconstriction in healthy and asthmatic human subjects (Pellegrino et al., J. Appl. Physiol. 81, 964-975, 1996). Once outside cells, ATP exerts different actions in various tissues and organs. These actions are mediated by distinct cell surface receptors, termed P2-purinoceptors. These receptors are different from the adenosine receptors, termed P1-purinoceptors. This distinction of different receptors is critical, as adenosine is a breakdown product of ATP. The P2-purinoceptors comprise two major families, P2X and P2Y. Each family consists of at least seven members (X1-7 and Y1-7). The P2X family represents cell membrane ligand-binding ion channels permeable to Na+, K+, and Ca2+. The P2Y-purinoceptors constitute G-protein-linked receptors, often coupled to phospholipase C and, hence, to inositol triphosphate formation. There are at least seven different subclasses of P2Y receptor, based upon agonist potency profiles. For a description of the various P2Y subtypes, see Abbrachio and Burnstock, Pharmac. Ther. 64, 445-475, 1994, the entire disclosure of which is incorporated herein by reference.
ATP has been shown to induce histamine release from rat peritoneal mast cells (Keller, Tissue Mast Cells In Immune Reactions, S. Karger, p. 38-39, 1966; Diamant, Int. Arch. Allergy 36:3-21, 1969; Sugiyama, Japan. J. Pharmacol. 21, 209-226, 1971; Cockcroft and Gomperts, J. Physiol 296, 229-243, 1979). One study attempted to identify the receptor which mediates the action of ATP on rat mast cells (Tatham et al., Euro. J. Pharmacol 147, 13-21, 1988). It was concluded in the study that the receptor is actually stimulated by a minor component of ATP, termed ATP4xe2x88x92 (Id.). ATP4xe2x88x92 effects are mediated through activation of the P2X7-purinoceptor (previously termed the P2Z-purinoceptor) expressed on the rat mast cell surface (Bennett et al., J. Physiol. (Lond.) 317:335-345, 1981).
While rat studies suggest that ATP can directly induce mediator release from lung mast cells, these results cannot necessarily be applied to human mast cells, as will be apparent from the following disclosure.
A method for inhibiting mediator release from stimulated human mast cells is provided. Human mast cells are contacted with an effective amount of an agent which inhibits ATP binding to P2-purinoceptors on the cells. Preferably, the agent inhibits ATP binding to a P2Y-purinoceptor on the cells, most preferably the P2Y1- or P2Y2-purinoceptor. The agent may comprise, for example, a P2Y-purinoceptor antagonist or an allosteric modifier of a P2Y-purinoceptor.
According to one embodiment of the invention, the stimulated mast cells so treated are mast cells which comprise immunologically stimulated mast cells. While the mast cells may be derived from any human tissue, the invention is most advantageously practiced on lung, gut or joint mast cells.
According to another embodiment, the invention is a method for treating a human subject for a disorder characterized by undesirable release of mediator from immunologically stimulated lung mast cells. An effective amount of an agent which inhibits ATP binding to P2-purinoceptors on mast cells is administered to the subject. The disorder may, for example, be a disorder characterized by the undesirable release of histamine, such as allergy or asthma. The disorder may also comprise inflammatory lung disease, or bronchoconstriction, such as bronchoconstriction associated with pulmonary embolism.
According to one particularly preferred embodiment of the invention, a human subject is treated for a bronchoconstriction caused by histamine release from stimulated lung mast cells by administration of an effective amount of an agent which inhibits ATP binding to a P2-purinoceptor, preferably to a P2Y-purinoceptor, most preferably the P2Y2- or P2Y2-purinoceptor, on lung mast cells.
The invention also provides a method for selecting agents useful for inhibiting mediator release from stimulated human mast cells. The method comprises contacting stimulated human mast cells with an agent which is an inhibitor of ATP binding to a P2-purinoceptor, preferably a P2Y-purinoceptor, most preferably the P2Y1- or P2Y2-purinoceptor; and assaying said cells for release of one or more mediators. The stimulated mast cells may comprise, for example, immunologically stimulated mast cells. Most preferably, the immunologically stimulated mast cells comprise lung mast cells. The preferred mediator for assay is histamine.
The invention is also a method for determining, in vitro, the effectiveness of an agent for the treatment of a human subject for a disorder characterized by undesirable release of mediator from stimulated mast cells. The method is a competitive binding assay in which the test agent competes with a P2-purinoceptor ligand for binding to a reagent comprising a P2-purinoceptor. The method comprises forming a mixture comprising the test agent, a P2-purinoceptor ligand (preferably a P2Y-purinoceptor ligand, most preferably a P2Y1- or P2Y2-purinoceptor ligand) and a reagent comprising a P2-purinoceptor (preferably a P2Y-purinoceptor, most preferably the P2Y1- or P2Y2-purinoceptor); and assaying the mixture for the inhibition of ligand binding to the receptor by the agent. The ligand preferably comprises a receptor agonist. The reagent may comprise, for example, human mast cells, particularly lung mast cells. The assay is particularly useful for determining the effectiveness of agents for the treatment of disorders characterized by the undesirable release of histamine, such as allergy and asthma.
By xe2x80x9cstimulated mast cellxe2x80x9d is meant a mast cell in an activated state which is characterized by, or proximally leads to, degranulation and release of mediator from the cell. By xe2x80x9cimmunologically stimulated mast cellxe2x80x9d is meant a mast cell which becomes stimulated by binding of antigen to IgE on the cell surface. Mast cell immunologic stimulation also includes experimental immunological stimulation achieved by contacting mast cells with antibodies to IgE, which results in the cross-linking of attached Fcxcex5R receptors on the mast cell.
By xe2x80x9cP2-purinoceptor ligandxe2x80x9d is meant a compound which binds to a P2-purinoceptor.